Genetic and Molecular in Vivo Analysis of Herpes Simplex Virus Assembly in Murine Visual System Neurons Jennifer H

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Genetic and Molecular In Vivo Analysis of Herpes Simplex Virus Assembly in Murine Visual System Neurons Jennifer H. LaVail,1* Andrew N. Tauscher,1 James W. Hicks,1 Ons Harrabi,1 Gregory T. Melroe,2 and David M. Knipe2 Departments of Anatomy and Ophthalmology, University of California San Francisco, San Francisco, California 94143-0452,1 and Program in Virology and Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 021152

Received 7 April 2005/Accepted 15 June 2005

Herpes simplex virus (HSV) infects both epithelial cells and neuronal cells of the human host. Although HSV assembly has been studied extensively for cultured epithelial and neuronal cells, cultured neurons are bio- chemically, physiologically, and anatomically signiﬁcantly different than mature neurons in vivo. Therefore, it is imperative that viral maturation and assembly be studied in vivo. To study viral assembly in vivo, we inoculated wild-type and replication-defective viruses into the posterior chamber of mouse eyes and followed infection in retinal ganglion cell bodies and axons. We used PCR techniques to detect viral DNA and RNA and electron microscopy immunohistochemistry and Western blotting to detect viral proteins in speciﬁc portions of the optic tract. This approach has shown that viral DNA replication is necessary for viral DNA movement into axons. Movement of viral DNA along ganglion cell axons occurs within capsid-like structures at the speed of fast axonal transport. These studies show that the combined use of intravitreal injections of replication- defective viruses and molecular probes allows the genetic analysis of essential viral replication and maturation processes in neurons in vivo. The studies also provide novel direct evidence for the axonal transport of viral DNA and support for the subassembly hypothesis of viral maturation in situ.

Herpes simplex virus 1 (HSV-1) infects mucous membranes sids are transported (19). However, not all capsids contain at sites of entry into the host. After infection of the epithelium, DNA. Some capsids, such as B capsids, may contain tegument the virus spreads to other cells in the epithelium and to the proteins, as well as histones and other immunoantigens, with- endings of sensory neurons, where it is taken up. The nucleo- out DNA (21, 37). Our understanding of viral-DNA transport capsid of the virus moves by retrograde transport within the kinetics and identification of the morphological compart- peripheral axons to the nuclear compartment of the sensory ment(s) in which it is transported have been hampered by the cell (for reviews, see references 32 and 36). Upon replication in lack of an adequate in vivo system for study. the neuronal cell body, new components of the virion move by Although HSV assembly has been studied extensively in anterograde transport from the neuron cell body to both pe- cultured cells and even more relevantly in cultured neurons, ripheral and central branches of the neuron (12). Envelope there are significant differences between cultured neurons and components, as marked by envelope-specific proteins, are mature neurons in situ. For example, mature uninjured verte- transported independently of capsid protein components, as brate axons in vivo have few ribosomes and few of the other marked by capsid-specific proteins (11, 27, 29). However, little factors necessary to produce proteins locally (5). In contrast, is known about the exact mechanisms of viral DNA transport embryonic neurites in vitro contain the cell machinery neces- or release from the axon. It has been assumed that viral DNA, sary for synthesis of new proteins (13). Theoretically, viral which is contained in reassembled envelope and capsid com- protein synthesis might also occur in situ within the neurites. ponents, must be released along the axon shaft and at axon Therefore, to fully understand HSV infection in mature neu- terminals, because the glial cells which envelop the infected rons, in vivo studies are necessary. axons also become infected with HSV (23, 39). To understand viral pathogenesis in neurons in vivo, our Previous models of HSV DNA transport are inferences strategy has been to use viral strains with known genetic mu- based on indirect evidence. First, morphological identification tations and defects in essential viral functions. However, the of capsids has been based on the presence of a dense core analysis of viral functions may be complicated because the within a small particle. However, uninfected sensory axons same functional defects may also prevent the spread of the contain granular vesicles that have dense cores and that are virus to the cell types of interest, e.g., neurons. For example, approximately the same diameter (75 to 95 nm) and morpho- replication-defective mutant viruses show little spread between logically similar to viral nucleocapsids. Second, staining of small vesicles (ϳ100 nm wide) within axons with a monoclonal cells or to sensory neurons, although nonessential functions antibody to VP5, the major capsid protein, suggests that cap- can be studied in vivo (16, 32, 39). Thus, while replication- defective mutant viruses are important tools for the study of viral gene function in culture, they have had fewer applications in vivo. * Corresponding author. Mailing address: Department of Anatomy, University of California San Francisco, San Francisco, CA 94143-0452. Recently, we developed a model of murine retinal ganglion Phone: (415) 476-1694. Fax: (415) 514-3933. E-mail: [email protected]. cell infection by directly injecting virus into the posterior cham-

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ber of the eye, thereby delivering virus to the retinal ganglion AGAATGTCTGTCC-3Ј, and the reverse primer was 5Ј-GATACAGGCCGGA cells and producing an infection (11). Current studies use this GTGTTGT-3Ј. Tissue isolation. At 2, 3, or 5 days postinfection (dpi), the animals were system to examine the infection of neurons in situ by replica- anesthetized, and each mouse was perfused intracardially with 20 ml of saline. tion-competent and replication-defective mutant HSV strains. The optic nerves, extending from the orbit to the optic chiasm (OC), were We tested whether injected virus was taken up by ganglion cells removed by following a standard protocol (11). In brief, the retinal ganglion cell and transported anterograde by a transcytotic mechanism. Sec- axons were dissected into four segments. The portion extending from the orbit to ond, we also described the viral structures that are involved in the OC was bisected into two segments, optic nerve 1 (ON1, 3 mm) and optic nerve 2 (ON2, 3 mm). The OC (2 mm) was dissected from the hypothalamus, and the axonal spread in viral DNA of an HSV infection in vivo. the optic tract (OT, 5 mm) was removed to the point where the axons enter the Last, we used electron microscopy (EM) immunohistochemis- dorsal thalamus. As a negative control, uninfected mice were prepared similarly. try, PCR, and EM in situ hybridization to document the fol- The same experiment was repeated three to four times for each time point. To lowing aspects of viral maturation and egress: (i) the rate of control for possible vascular transfer of virus, we took samples of cerebellum from infected mice; we failed to find any viral DNA in these control samples. transport of viral DNA in the axon and (ii) the identity of the Aliquots of viral stocks were used as positive controls. structure within which viral DNA is transported. Polyacrylamide gel electrophoresis and Western blotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with antibodies to VP5, the major capsid protein, and to gD, one of the major viral envelope MATERIALS AND METHODS proteins, were performed as described previously (11). Equivalent amounts of protein were loaded for each lane. When we omitted the primary monoclonal Antibodies and culture supplies. Monoclonal antibody (gD-1D3) specific for antibody in Western blotting and incubated in the secondary antibody only, i.e., HSV-1 glycoprotein D (gD) was provided by Gary Cohen and Roselyn Eisen- sheep anti-mouse antiserum, we consistently found a band at ϳ55 kDa in all berg, University of Pennsylvania, Philadelphia. An additional monoclonal anti- lanes. This represents the nonspecific binding of the second antibody to the body specific for HSV-1 gD (MAB8684) was purchased from Chemicon Inter- heavy chain of the immunoglobulin G reacting to the mouse. When we omitted national, Temecula, CA. Two monoclonal antibodies specific for the HSV major the second antibody, we saw no bands on the immunoblots. capsid protein ICP5 were also obtained (no. C050IM [Biodesign International, DNA preparation and PCR procedures. We pooled tissue from 10 eyes to Saco, ME] and ICP5 [Virusys Corp., North Berwick, ME]). Polyclonal rabbit normalize the amount of viral DNA for each treatment condition and time point. anti-HSV–horseradish peroxidase (HRP) (no. AXL298P) and rabbit anti-digoxi- Genomic DNA from the nerve specimens was isolated as previously described genin (DIG)–HRP (no. AXL5227P) were from Accurate Chemical and Scien- (25). The resulting pellet was suspended in Tris-EDTA, and the DNA concen- tific, Westbury, NY. Sheep anti-mouse immunoglobulin G–HRP (NA-931) was tration was determined by A260 measurement. One hundred nanograms of DNA from purchased Amersham Biosciences, United Kingdom. For protein assays, we was used for the PCR analysis. To determine the limit of detection, we serially used the BCA kit and protocol from Pierce (Rockford, IL). All culture supplies diluted 10-fold the PCR product made using the gD primers, starting at 100 ng. were obtained from the Cell Culture Facility, UCSF. The lower limit of detection was ϳ1 fg of DNA, which is the equivalent of 3,400 Preparation of viral stocks. African green monkey kidney (Vero) cells were copies of DNA. grown and maintained as described previously (11). Vero cells were infected at For PCR analysis, forward and reverse primers targeting the Us6 (gD)se- a multiplicity of infection of 0.01 with the HSV wild-type (wt) KOS strain. After quence of HSV-1 were used. Primer sequences are 5Ј-ATGGGAGGCAACTG 48 h, 100% of the cells showed cytopathic effects. Progeny virus was harvested by TGCTAT-3Ј for the gD forward primer and 5Ј-CTCGGTGCTCCAGGATAA pooling cells and medium and freeze-thawing three times. Cellular debris was AC-3Ј for the gD reverse primer. These primers yield a gD-specific PCR product ␮ removed by filtration through a 0.45- m sterile filter (Millipore, Billerica, MA). of 250 bp. Twenty-microliter PCR mixes contained the following: 100 ng DNA, The filtrate was centrifuged at 25,000 rpm in a Beckman SW28 rotor for2hat 1ϫ QIAGEN PCR buffer (final MgCl concentration, 1.5 mM), 0.15 ␮mgD 4°C through a step gradient of 10%, 30%, and 60% sucrose. Virus was collected forward primer, 0.15 ␮m gD reverse primer, 5% dimethyl sulfoxide, 0.2 mM from the 30% to 60% interface, suspended in phosphate-buffered saline (PBS), deoxynucleoside triphosphates, and 0.5 U Hot Star polymerase (QIAGEN, Va- and collected by centrifugation for1hat4°C. The pellet was suspended in lencia, CA). PCR mixes were subjected to thermal cycling under the following Ϫ minimal essential medium without serum and stored at 80°C. The viral titer conditions: 15 min at 94°C and 25 cycles of 94°C for 30 s/52°C for 30 s/72°C for was determined by a standard plaque assay. 1 min. A final extension reaction was carried out at 72°C for 6 min. The PCRs Two other HSV preparations were used in this study. We exposed aliquots of were run on a 1% agarose gel and stained with ethidium bromide according to the wt strain to UV light at a 254-nm wavelength to inactivate viral infectivity standard procedures (31). Aliquots of wt and ICP8Ϫ stocks of HSV were used as (33). After 20 min of exposure, the titer dropped 1,000-fold. We used this positive controls. Relative estimates of the amounts of viral DNA were obtained UV-treated KOS (KOS-UV) as a control for virus that is replication competent. by using NIH ImageJ to quantify the density of bands in the films. The HSV-1 KOS UL29 gene mutant, 8lacZ, was constructed by insertion of an EM immunohistochemistry. The mice were anesthetized, perfused, and dis- ICP8-lacZ fusion cassette into the ICP8/UL29 gene, as described previously for sected as previously described (11). The segments of the optic path were treated HSV-1 KOS1.1 (4). This viral strain is capable of expressing immediate early, for EM immunohistochemistry by using an HRP-tagged polyclonal antiserum early, and some late viral gene products but is replication defective in normal against human HSV according to standard procedures (22). In some experi- cells. ments, the segments were treated with a monoclonal antibody to ICP5 (Virusys Intraocular injection. All procedures involving animals adhered to the Dec- Corp.) at a dilution of 1:100 overnight, followed by use of a mouse-to-mouse laration of Helsinki and the Guiding Principles in the Care and Use of Animals detection kit (no. 2700; Chemicon), according to the instructions of the manu- and to the guidelines of the UCSF Committee on Animal Research. Eyes of male facturer. Thin sections for EM were not counterstained with uranyl acetate or BALB/c mice (5 to 6 weeks of age) were infected with equivalent titers of virus lead citrate. When the primary antibody was omitted, we found no immuno- in 4 ␮l of sterile minimal essential medium without serum at concentrations of stained structures in the retinal ganglion cells or glial endfeet (data not shown). ϫ 4 ␮ approximately 9 10 PFU/ l, according to a standard procedure (11). We Production of in situ probe. Primers for Us6, as described above, were used to introduced valacyclovir hydrochloride (1 mg/ml) (Valtrex; Glaxo Wellcome, Inc., prepare the PCR product for DIG labeling. Approximately 4 ϫ 105 PFU per Greenville, NC) into the drinking water 24 h after ocular infection to block reaction of wt virus was used as template DNA in 50-␮l reaction volumes further replication (11). This infection protocol allows a restricted time period of containing 1ϫ Hot Star (QIAGEN) buffer, 5% dimethyl sulfoxide, 2 mM de- viral replication in retinal ganglion cells and interferes with replication in astro- oxynucleoside triphosphates, 0.15 ␮M of each primer, and 1.0 U Hot Star Taq cytes in the optic nerve (11). DNA polymerase. Cycling conditions were as follows: 15 min at 94°C and 40 Retinal mRNA analysis. Total RNA was purified from infected mouse retinas cycles of 94°C for 30 s/52°C for 30 s/72°C for 1 min. A final extension reaction was by using the Trizol reagent and protocol (Invitrogen Corp., Carlsbad, CA). The carried out at 72°C for 6 min. Ten of these PCRs were pooled and purified using integrity of the mRNA was verified using primers to mouse GAPDH (glyceral- a QIAquick PCR purification kit (QIAGEN). The final volume was 10% of the dehyde-3-phosphate dehydrogenase). The RNA was reverse transcribed into original pooled volume. Quantitation was estimated by running 1 ␮lofthe cDNA by using random hexamer primers according to the Invitrogen Superscript purified PCR product on a 1% agarose gel with subsequent ethidium bromide first-strand synthesis system for reverse transcription (RT). The resulting cDNA staining. The intensity of the band was compared to that of the stained standard

from the RT reaction was tested for the presence of Us8 (gE) by using gE DNA. The integrity of the PCR product was veriﬁed by sequencing analysis gene-speciﬁc primers in a PCR (6). The gE forward primer was 5Ј-AGCTCCC (Biomolecular Resource Center, UCSF). An aliquot (300 to 600 ng) of each PCR 11144 LAVAIL ET AL. J. VIROL.

FIG. 2. (A and B) Viral mRNA was detected in retinal extracts at

24 h postinfection. mRNA of Us8 (gE) was found in retinas in the wt (lane 1) or in the ICP8Ϫ (lane 3) strain of HSV. There was no expression in retinas infected with the KOS-UV-treated strain (lane 2) or in FIG. 1. (A and B) Viral DNA was detected in retinal extracts at cerebellar tissue from mice infected with the wt viral strain (lane 4). 24 h postinfection. Bands migrating at approximately 219 and 250 bp (B) The GAPDH mRNA internal control was positive.

indicate the presence of Us8 for gE (A) and Us6 for gD (B) DNA, respectively. We detected DNA fragments in wt-infected (lane 1) and ICP8Ϫ-infected (lane 2) retinas. There was no viral DNA detected in uninfected eyes (lane 3) or in eyes injected with cocktail alone (lane 4). Characterization of retinal infections. In these studies, we Positive controls included DNA from the wt (lane 5) and ICP8Ϫ (lane used PCR to measure viral-DNA movement in axons in vivo. 6) viral stocks. We first determined that the assay was sensitive enough to detect input viral genomes. We infected mice with wt or ICP8Ϫ virus and, 24 h later, euthanized the animals. The posterior eye was dissected and DNA was isolated. To determine if we could product was labeled with DIG-dUTP by using a DIG DNA labeling kit from Ϫ Roche and following the manufacturer’s protocol. The labeled probe was quan- detect viral genomes from the ICP8 mutant virus in the ret- titated using a DIG nucleic acid detection kit from Roche. ina, we used primers for gE and gD gene sequences for PCR EM in situ hybridization. Five days after inoculation, mice were anesthetized amplification. We detected both DNA sequences in wt virus- and perfused according to standard procedures (11). The tissues were fixed for infected eyes and, to a lesser degree, in ICP8Ϫ virus-infected 24 h to 3 weeks at 4°C, and the optic nerves and tracts were dissected and cut manually into thin slices. We modified the procedure described by Hayat (7) as eyes (Fig. 1A and B, lanes 1 and 2). Based on densitometric follows. The tissues were rinsed three times for over 30 min in PBS, incubated in scans of the gel, about sixfold more of the Us8 gene sequence 10 mM sodium citrate (pH 6.0), and then heated to boiling for 1 min. The and about two- to fivefold more of the Us6 sequence were sections were incubated in 1.0 M sodium thiocyanate for 10 min at 80°C and then detected after infection with wt virus than after infection with washed two times for 5 min each in distilled water. The sections were incubated Ϫ at 78°C for 10 min to denature the DNA and then incubated overnight at 37°C the ICP8 , as a result of the impaired DNA replication of this in the hybridization buffer. This buffer was composed of the following: deionized strain. No DNA was detected in uninfected eyes or in the formamide (65%), 2ϫ SSC buffer (0.3 M sodium chloride and 0.03 M sodium cocktail-injected eyes. Thus, our PCR could detect input viral citrate), 10% dextran, salmon sperm DNA (1 ␮g/␮l), and DIG-labeled probe (40 genomes. ␮ l at 20 ng/ml). To further test that the viruses introduced into the eye were The tissues were rinsed two times for 10 min each in 2ϫ SSC buffer with 50% formamide at 40°C and rinsed three times for 5 min each in PBS. To block capable of infecting retinal cells, we infected mice with the wt, Ϫ endogenous peroxidases, the tissues were rinsed in 0.3% hydrogen peroxide for KOS-UV, or ICP8 mutant strain and euthanized the mice at 15 min and rinsed in PBS. To block nonspecific binding, the samples were 24 h postinfection. The posterior portions of the eyes were incubated in 4% normal rabbit serum in PBS for1hatroom temperature. After dissected, retinal RNA was purified and subjected to RT, and a rinse in PBS, the tissue was incubated overnight at room temperature in HRP-tagged rabbit anti-DIG antiserum. The tissue was washed three times for the resulting product was amplified by PCR with primers for ␥ 5 min each in PBS and processed for EM immunohistochemistry according to Us8 (gE), a -1 or early/late viral gene (30). We measured the standard procedure (22). Thin sections were not counterstained. Positive con- expression of a ␥-1 or leaky late gene, because ␥-1 gD was one trols included optic tracts from wt virus-infected animals that were not treated of the proteins assayed for transport in axons. Both wt virus- with valacyclovir. We found labeled viral DNA in capsids in astrocyte nuclei infected and ICP8Ϫ virus-infected retinas expressed gE mRNA throughout the optic pathway (data not shown). Negative controls included tissue from uninfected animals, tissue from infected animals in which the primary (Fig. 2, lanes 1 and 3). Neither KOS-UV nor control (unin- antibody was omitted, and tissue from infected animals in which the DIG probe fected) retinas expressed gE mRNA (Fig. 2, lanes 2 and 4). was omitted. We found no in situ staining in any of the negative controls. GAPDH mRNA was assayed as a positive control. These results indicated that wt and ICP8Ϫ strains were capable of RESULTS infecting retinal tissue by 24 h after injection. To define the cellular localizations of the wt and ICP8Ϫ We wished to determine the role of viral-DNA replication in viruses in retinal ganglion cells, we examined retinal tissues viral assembly and axonal transport in murine visual system after EM immunohistochemical staining. Retinal ganglion cells neurons in situ following injection of the HSV strains into the are the sole source of axons to the optic nerve. Although posterior chamber of the eye. Our first step was to characterize potential amplification of virus might also occur in non-gan- the infection model and the assays used to detect viral DNA, glion cells, these cells cannot contribute to viral transport into RNA, and proteins. the optic pathway, because they do not have processes that VOL. 79, 2005 AXON TRANSPORT OF HSV DNA IN VIVO 11145

FIG. 3. A variety of particles were immunopositive in retinal ganglion cell bodies 24 h after infection with the wt (A, C, and D) or the ICP8Ϫ (B) strain of HSV. (A) HSV immunostaining coats the inner limiting membrane of the retina (arrow) and the surface of Mu¨ller glial endfeet. Various immunopositive particles occupy the ganglion cell cytoplasm. One immunopositive particle (asterisk) is located in close proximity to the nucleus. Bar ϭ 600 nm. (B) Labeled particles are also found in the cytoplasm of ganglion cells infected with the ICP8Ϫ virus. A magniﬁed image is shown in the inset. The inner limiting membrane is interrupted in some sites (arrow). This could allow access of virus to the neuronal cell surface and eliminate the usual requirement that the virus pass through the glial cell before reaching the neuron. Bars ϭ 300 nm (B) and 100 nm (inset). (C) A virion lies adjacent to the Mu¨ller glial endfeet of the inner limiting membrane. The endfeet are sealed at a junctional complex (arrow). Bar ϭ 200 nm. (D) Capsids (asterisks) are clustered in a ganglion cell axon in the optic ﬁber layer of the retina. An unlabeled, dense core vesicle is also present (arrow). Bar ϭ 200 nm. a, axon; g, glial endfoot; n, nucleus.

extend beyond the retina. Thus, we focused on infection in imal portion of ganglion cell axons in the ganglion cell layer of retinal ganglion cell bodies. the retina also contained a variety of immunopositive or- At 24 h postinfection with wt virus, we found evidence of ganelles, including some that were about 110 nm in diameter HSV antigens at the vitreal surface and on the surface of (similar in size to capsids) and some that were signiﬁcantly Mu¨ller glial cell endfeet that are extensions of the cytoplasm of larger (Fig. 3D). Dense cored vesicles about 110 nm in diam- the Mu¨ller cells, the major glial cell type of the retina (Fig. 3A eter that were not immunopositive were also noted in the axon and C). In retinal ganglion cell bodies, we found a variety of (Fig. 3D). At 24 h after infection with the ICP8Ϫ mutant virus, immunopositive vesicles and nucleocapsids, including one that we found less intense immunoreactive product coating the was located adjacent to the nucleus (Fig. 3A). The most prox- inner limiting membrane of the retina and the surface of Mu¨l- 11146 LAVAIL ET AL. J. VIROL.

FIG. 4. The axonal transport of the wt proteins was assayed by Western blotting of segments of the optic pathway. The envelope protein, gD (left panel), was detected in the ON1 and ON2 segments of the optic pathway at 3 dpi. The capsid protein, VP5 (right panel), was detected throughout the pathway by 3 dpi. gD was detected in ON, ON2, and the OC by 4 dpi. VP5 persisted throughout the pathway. (ϩ), viral stock aliquots; (Ϫ), optic pathway homogenates from uninfected animal. Markers ϭ 50 kDa (left panels) and 155 kDa (right panels). ler glial cell endfeet (Fig. 3B). We were unable to identify any infected retinal ganglion cell bodies after injection with FIG. 5. The axonal transport of viral DNA was assayed for PCR KOS-UV virus. product primed off of Us6. (A) Comparison of the transport of viral Replication is necessary for viral transport. We tested DNA from different viral strains to the OT segment 2, 3, or 5 dpi. whether injected virus was taken up by ganglion cells and Positive bands were found in the OT from animals infected with wt virus at 3 and 5 dpi, but no DNA was found in the tracts of mice 2, 3, transported anterograde by a transcytotic mechanism. This Ϫ process is defined as delivery of a protein or complex from one or 5 days after ICP8 or KOS-UV infection. The cerebellum (CB) from the wt was uninfected. (B) Translocation over time of wt DNA in pole of a cell to the other pole of the cell, via vesicle-mediated, axons. Viral DNA was detected in the proximal (ON1 and ON2) optic fast axonal transport, and by this mechanism, no DNA repli- pathway 48 h after infection. By 60 h, the DNA was detected in all four cation would be necessary. Thus, if a replication-defective segments and persisted for an additional 12 h. HSV is anterograde transported, then transcytosis could be a mechanism for viral targeting to the axon. After retinal ganglion cell infection, we used Western blots HSV. There was no evidence of viral DNA in tissue from the and antibodies to VP5 and gD, markers of the capsid and cerebellum, which was assayed as a control for possible sys- envelope subassemblies, respectively, to determine that the temic or viremic delivery. These findings confirm that replica- viral proteins were synthesized and transported in the ganglion tion of new virus is essential for the transport of viral DNA to cell axons. By 3 dpi with wt virus, we found gD in the ON1 and the axon and that transcytosis of DNA from the original in- ON2 segments (Fig. 4). We detected VP5 in ON1, ON2, and jected viral stock does not play a role in anterograde transport. the OC in two of three experiments at 3 dpi; in a third exper- Temporal course of transport of wt viral DNA. By 48 h after iment, we identified VP5 as far as the OT (Fig. 4). By 4 dpi, we infection, viral DNA was detected in the ON1 and ON2 seg- detected gD in ON1, ON2, and the OC and VP5 throughout ments of the optic nerve that are closest to the retinal ganglion the pathway. Thus, we found that the transport of gD was cell bodies (Fig. 5B). By 60 h, we detected viral DNA in these somewhat slower than that of VP5. This finding differs from segments and in the remaining portions of the optic nerve. The the result we obtained earlier, in which we found that gD was viral DNA was concentrated about three- to fivefold greater in detected in the axon of retinal ganglion cells infected with the ON1 than in ON2 at 48 and 60 h after infection (Fig. 5B). By F strain of HSV at least a day earlier than the VP5 protein 72 h, it was more homogenously distributed throughout the could be detected (11). In the current study, we used the KOS pathway. Assuming that the front of DNA transport travels a strain, the background strain of the ICP8Ϫ mutant strain. distance of about 6 mm (from the OC to the length of the OT) When we injected ICP8Ϫ or KOS-UV virus, we found no VP5 in 12 h (from 48 to 60 h postinfection), this result suggested a or gD proteins in Western blots of optic pathways from ani- transport rate of ϳ0.14 ␮m/sec. This rate was consistent with mals at 3, 4, or 5 dpi (data not shown). These results indicate rapid axonal transport. that viral replication is a necessary step for the expression, The rate was inconsistent with spread from glial cell to glial targeting, or axonal transport of viral proteins. cell, outside of retinal ganglion cell axons (1, 23). There are We used PCR to measure the viral-DNA content in each about 2.4 astrocytes/33-␮m length of optic pathway; the length section of the optic pathway, to assay the rate of DNA trans- of the pathway from ON1 to the OC is about 6 mm (11, 15). A port, and to test whether viral replication was necessary for chain of about 465 astrocytes would be needed to span the DNA delivery to the mouse optic tract. In contrast to the distance from the optic nerve head to the beginning of the OC. appearance of viral DNA in the ICP8Ϫ-infected retinas (Fig. To reach the beginning of the OC by 48 h, the HSV would have 1), no viral DNA was detected in the optic tracts at any time only about 6.2 min/astrocyte to enter, replicate, and spread to point. No evidence of a PCR product for the Us6 gene (gD) was the next astrocyte. found in the optic tracts of either ICP8Ϫ-infected or KOS-UV- EM localization of viral protein and DNA. In the most infected mice at 2, 3, or 5 dpi (Fig. 5A). However, a PCR proximal portion of ganglion cell axons, we found a variety of product was found in the optic tracts at 3 and 5 dpi with wt organelles containing viral antigens (Fig. 3D) (see above). In VOL. 79, 2005 AXON TRANSPORT OF HSV DNA IN VIVO 11147

FIG. 6. EM immunohistochemistry of cross-sections of axons in the optic tracts of mice 5 dpi with wt strain. Two types of HSV-positive particles were seen in retinal ganglion cell axons (a). (A) Small (ϳ110-nm-wide), round, immunostained particles (enlarged in upper inset). Bars ϭ 1 ␮m (A) and 200 nm (inset). (B to D) Larger membrane-bound organelles surrounded an immunostained substructure. Bars ϭ 400 nm. the most distal portions of the same axons, i.e., in the optic axons from infected mice that had been hybridized with a tracts, we found two sizes of immunostained particles at 5 dpi. DIG-labeled probe for the Us6 (gD) gene (Fig. 7C through F). One class of particle was about 150 to 180 nm in diameter and Negative controls included tissue from uninfected mice or tis- was composed of a host membrane surrounding viral antigenic sue from infected mice in which the primary antibody or DIG contents (Fig. 6B through D). label was omitted. Similar structures were never observed in The other class of particles was small (ϳ110 nm in diame- the control tissues (data not shown). These results demon- ter), capsid-like, and often had a diffuse halo surrounding the strate directly that the major structure in which viral DNA is particle (Fig. 7A). The capsid-like particles resembled struc- axonally transported is a nucleocapsid. tures immunostained with a monoclonal antibody to VP5 that were found within the nuclei of infected astrocytes distributed DISCUSSION along the OT in tissues from animals not given valacyclovir (Fig. 7B). Localization of viral DNA within the capsid-like Genetic analysis of HSV assembly in situ. We have used a structures was conﬁrmed by examination of sections of optic genetic approach to test if viral-DNA replication is needed for 11148 LAVAIL ET AL. J. VIROL.

virus in each cell body in a synaptic chain. Our studies dem- onstrate that viral-DNA replication must occur for HSV to move to and be transported anterograde down the length of the axon in detectable amounts. Virus that is incapable of replication, as a result of either UV irradiation or genetic mutation, fails to be delivered to the axon compartment. Several explanations might account for why mutant virus is not detected in the optic pathway. The concentration of virus that reaches the axon compartment may be too low to be detectable. The cellular-transport machinery of an infected neuron may not be able to recognize nucleocapsids that enter the retinal ganglion cell. Alternatively, the host machinery may be able to recognize nucleocapsids, but the nucleocapsid itself may lack the necessary recognition signals. Whatever the fail- ure of recognition, transcytosis of virions does not play a significant role in the delivery of HSV DNA to the axon. The recognition mechanism that allows encapsulated viral DNA to be sorted to the axon compartment remains unknown. New nucleocapsids are associated with a complement of tegument proteins which may provide some sort of signal for recognition between the viral particle and the motor-associated transport proteins (see below) (for a review, see reference 18). Diefenbach et al. have suggested that conventional kinesin heavy chain and the US11 tegument protein are associated during capsid movement (3). Rate of movement of viral DNA in the axon in situ. We and others have previously tried to identify particles found at the FIG. 7. Immunohistochemical and DNA in situ labeling of the small, round particles in the optic tracts of mice 5 dpi. (A) Polyclonal EM level in infected axons as enveloped virons (2, 9, 12, 23). antiserum to human HSV labeled a small particle in an axon, similar to We have faced the difficulty of distinguishing between dense that illustrated in Fig. 6A. (B) Similar particles are immunolabeled core vesicles which are characteristic of sensory and autonomic with a monoclonal antibody to VP5 in the cytoplasm of an infected neurons and viral particles (28). Without the advantage of astrocyte in the OT. (C to F) The DIG-HRP reaction product is immunostaining, this is problematic; the vesicles and particles concentrated on similar small, round particles in the axons. Bars ϭ 200 nm. are about the same size, and both contain an electron dense core. EM immunostaining with antibodies to viral proteins and DNA probes resolve the difficulty; viral particles are stained, and dense core vesicles are not. viral DNA to enter the axon in an infected neuron in situ. In this study, we also estimated the rate of transport of HSV Because viral-DNA synthesis is absolutely necessary for viral DNA in mature, infected central nervous system neurons in replication, viruses defective for DNA synthesis are unable to vivo. Specifically, we found HSV DNA in the most distal ax- spread from cell to cell (32). Thus, we needed to deliver these onal segment (the OT) by 2.5 dpi. Viral capsid protein VP5 viruses directly to the host neuron by injection. Although pre- was detected in this distal optic segment at 3 to 4 dpi. This vious studies have used viruses that are defective for nones- difference is likely due to the difference in sensitivities of PCR sential gene products, such as viral glycoproteins that deter- for viral DNA and Western blotting for viral protein, although mine neuronal targeting in vivo (38), this is the first attempt to other explanations are possible. examine the role of an essential viral-replication process in An estimate of the rate of axonal transport of the viral DNA viral assembly in vivo through the use of replication-defective can be made if one makes several assumptions. First, we as- mutants. Despite the presence of viral particles around retinal sume that the viral DNA is transported at about an equivalent ganglion cell axons in the optic fiber layer, these replication- rate in all infected axons. Potential variability is reduced but defective virions failed to reach the proximal portions of the not eliminated by valacyclovir treatment, which limits the pe- optic nerve. riod of infection to the first 24 h. Second, we assume that the Neurons can internalize a variety of macromolecules, includ- average length of the nerve between the OC to the point where ing trophic factors, lectins, toxins, and other pathogens. After the axon reaches the lateral geniculate nucleus of the thalamus uptake at the dendritic or cell soma membrane, these macro- is about 6 mm. Third, we assume that the appearance of DNA molecules can be moved into and down the axon. For example, in a segment is not limited to the most proximal limit or the wheat germ agglutinin is transcytosed by chick retinal ganglion most distal limit of that segment. Given these assumptions, we cells (10). Subsequent release after anterograde transport re- found that DNA is transported about 6 mm in 12 h (between sults in transcytosis across the neuron and release at the syn- 48 and 60 h postinfection). This suggests that the viral DNA apse (for a review, see reference 40). It has not been clear moves at a rate of about 0.5 mm/hr or 0.14 ␮m/sec. whether viruses can be similarly transcytosed across a neuron This estimated rate is less than 10% of the rate estimated for or whether transneuronal transfer occurs by the replication of instantaneous axonal transport of viral capsids in embryonic VOL. 79, 2005 AXON TRANSPORT OF HSV DNA IN VIVO 11149

immunopositive for HSV. The surrounding membrane did not react with the HSV antiserum. The identity of these organelles remains to be determined, and the use of monoclonal antibodies should clarify the particular viral proteins that are intrinsic to the particles. One possibility is that the structures are late endosomal organelles that have picked up viral proteins at the cell surface and are in the process of retrograde transport from the axon surface back toward the cell body (26). However, since we see no immunolabel on the axon surface, this seems unlikely. Alternatively, they may contain newly synthesized viral envelope components. Mettenleiter has proposed a model in which trans-Golgi vesicles partially envelop nucleocapsids in the cytosol (17). The glycoproteins within the trans-Golgi vesicle may be segregated to one pole of the vesicle by interaction FIG. 8. Model of derivation and axonal transport of two classes of with proteins of the tegument that surround the nucleocapsid. particles. In the neuron cell body, a membrane vesicle derived from the The portion of the vesicle that contains viral glycoproteins trans-Golgi network and containing envelope proteins partially enfolds invaginates into the lumen, resulting in a double-membrane a maturing capsid and associated tegument proteins (asterisk). In the cell body-proximal axon, the capsid is separated from the vesicle (de- vesicle: the host cell membrane surrounding the viral envelope noted by the number 2). The capsid containing the DNA is transported membrane (Fig. 8). The capsid is transported independently of independently of the host cell vesicle containing envelope components the vesicle (Fig. 8). The union of the capsid and envelope (denoted by the number 1). N, nucleus; G, Golgi cisternae; TGV, components in the axon may occur by budding of capsid, DNA, trans-Golgi vesicle. and tegument into the larger immunopositive organelle that we have found (39). However, viral egress from an axon may depend on more than one mechanism, and our sample is small. neurons in vitro, which is about 1.97 ␮m/sec and with an In summary, after the entry of HSV into neurons and the average run length of about 13 ␮m (34). Several factors can delivery of viral DNA to the neuronal nucleus, new viral com- account for this difference between in vivo and in vitro esti- ponents and DNA must be produced before the viral DNA mates. First, the in vitro estimates are done for single particles moves anterograde from the cell soma to nerve terminals in moving small distances interrupted by pauses. The pauses be- quantities we can detect. A critical question has been the tween movements were not included in the rate of movement. identity of the package in which the DNA is transported within Our in vivo measurements are done for populations of parti- the axon. Using EM immunohistochemistry, we identified two cles moving over significantly longer distances, and most mea- types of HSV-immunopositive organelles in the OT by 5 dpi: surements certainly include the time of delays between salta- small, round organelles and larger, membrane-bound vesicles. tory jumps and temporary reversals in direction. Second, the Both contain viral proteins, as evidenced from the immuno- difference in rate in vivo may relate to the steric interference of staining. Using EM in situ hybridization, we provided new moving DNA (in capsid) with other organelles in axons of evidence that the smaller, ϳ100-nm capsid-like structures con- larger caliber. Last, the composition of axoplasm in mature tain viral DNA. These results provide the first definitive evi- axons and the stability of microtubules in mature axons are dence for viral-DNA transport in the capsids. They provide known to be different than those of relatively immature neu- support for the subassembly model of viral maturation in situ rites (24). and show that movement of viral DNA into the axon requires Structures containing viral DNA in the OT. By 5 dpi, we new synthesis of viral DNA. found viral proteins localized in at least two types of particles ACKNOWLEDGMENTS in the axons of the OT: small, round 110-nm particles and larger 150- to 180-nm particles. The smaller, round particles This work was supported by PHS grants P01 NS35138 (D.K.), P01 were immunopositive with a polyclonal antiserum to HSV and HL-24136, EY-08773, and P30 EY02162 (J.L.) and by funds from That positive for viral DNA by EM in situ hybridization. The capsid Man May See, Inc., and Fight for Sight. 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